Nucleic acid extraction kit, nucleic acid extraction method, and nucleic acid extraction apparatus

ABSTRACT

A nucleic acid extraction kit, which enables the nucleic acid extraction operation to be accomplished safely without causing contamination, and in which the complex preparation of reagents and the disposal treatments that are performed before and after the nucleic acid extraction operation can be performed rapidly and simply, with the extraction performed in an automated manner. The nucleic acid extraction kit includes: a container including reagent wells that each store at least a reagent, a sample well into which a biological sample is introduced, a waste liquid well, and a collection well in which an extracted nucleic acid is collected, and an extraction filter cartridge equipped with an extraction filter for separating and extracting a nucleic acid from the biological sample, wherein the extraction filter cartridge is formed in a manner that enables the extraction filter cartridge to be supported on the waste liquid well and the collection well.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/381,817 filed Dec. 30, 2011, which claimed the benefit under 35U.S.C. Section 371, of PCT International Application No.PCT/JP2010/058557, filed May 20, 2010, which claimed priority toJapanese Application Nos. 2009-162588 and 2009-162589, filed Jul. 9,2009 in the Japanese Patent Office, the disclosures of which are herebyincorporated by reference.

TECHNICAL FIELD

The present invention relates to technology for conducting pretreatmentsin a method and apparatus used in genetic analysis for concentrating andpurifying a nucleic acid extracted from a biological sample and thenperforming a genetic analysis treatment. Further, the invention relatesto a nucleic acid extraction kit that enables the sample supply sourceand the disposables generated by sample treatment to be discarded duringthe pretreatment steps without causing contamination, and also relatesto a nucleic acid extraction method that uses this extraction kit.

BACKGROUND ART

In recent years, trials have been conducted of introducing geneticanalysis into medical settings and the like. These trials have involvedtesting and analysis of nucleic acids extracted from biological samples.Biological samples refer to various bodily fluids such as blood, plasma,serum, semen and lymphatic fluid, various tissues such as skin, hair andmuscle tissue, and various excreted substances such as feces and urineobtained from a target living organism (or the corpse of a livingorganism), as well as other samples derived from the above samples.Testing of these biological samples offers the possibility of rapiddetection of diseases and their pathogenic risk, as well as earlydetection of infectious diseases and malignant tumors.

Extraction of a nucleic acid from a biological sample is an importantprocess in the fields of genetic engineering and clinical testing. Forexample, in those cases where a blood or tissue sample is collected froma human body and subjected to clinical testing, only the nucleic acidmust be extracted from the biological sample such as the blood sampleand then purified. In order to separate the protein and the nucleic acidfrom the other components, the biological sample is first subjected to aphysical or chemical dissolution treatment, and an extraction operationis then used to separate the protein and fats from the nucleic acid,thereby extricating the nucleic acid. During this operation, the processof separating and purifying the protein and the nucleic acid is prone tosample contamination. Organic solvents are widely used in standardextraction methods, but suffer from a number of problems, includingtoxicity of the solvent itself, the time and effort associated withtreating discards, the time and effort associated with centrifugalseparation operations, and the possibility of contamination of thesample or infection from the sample.

The BOOM method is one known method for extracting and collecting anucleic acid. The BOOM method is a nucleic acid extraction techniquethat combines a chaotropic agent and a solid-phase silica or the like,and utilizes adsorption of the nucleic acid on the silica surface in thepresence of chaotropic ions.

In conventional methods, the disruption and extricating step, and theextraction and separation step are performed separately, and because thenucleic acid separation and extraction cannot be performed as part ofthe pretreatment, the possibilities of infection from the sample andcontamination of the sample always exist between the two steps.Moreover, because these operations require human involvement, they tendto be inefficient and complex, and there is a possibility that operatorsmay become infected with pathogens.

One example of an apparatus that uses a solid-phase extraction filter isthe nucleic acid extraction apparatus disclosed in Patent Document 1. InPatent Document 1, an integrated column having a plurality of columnsarranged in a two-dimensional manner is used to enable a plurality ofsamples to be treated simultaneously. A vacuum chamber is formed byinstalling this integrated column (extraction filter cartridge) in anairtight manner at an upper opening of a vacuum chamber, enabling asolid-phase extraction to be performed using the so-called suctionmethod. In those cases where a nucleic acid is to be extracted, theseries of steps of adsorbing the nucleic acid to a carrier, washing thecarrier, and then desorbing and collecting the nucleic acid from thecarrier must be performed.

Further, a technique for independently attaching containers (wells)containing the various reagents used for extracting the nucleic acid, acollection container and a waste liquid container to an apparatus hasalso been proposed (see Patent Document 2). The nucleic acid extractionapparatus and cartridge disclosed in Patent Document 2 enable treatmentto be performed without complex reagent dispensing operations, simply byadding the sample to the apparatus, and is therefore ideal for treatinglarge amounts of a plurality of samples.

However, with the techniques disclosed in the above Patent Documents,considerable time and effort is required in preparing the extractionfilter cartridges, collection tubes and waste liquid tubes and the like,and even installing these items on the apparatus requires a significantamount of time. Moreover, in these apparatus, there is possibility ofcontamination of the surroundings before and after operation of theapparatus. In other words, in the nucleic acid extraction process, aplurality of items such as the integrated column or the variouscontainers and the like must be installed on the apparatus and thenremoved following use, and therefore there are significant possibilitiesof splashing of the waste liquid due to vibrations or the like, or foamformation depending on the nature of the solutions, resulting incontamination of the extracted sample. Furthermore, during the disposalprocess, there is a danger that overturning or deviation of the wasteliquid tube installed in the apparatus may cause contamination.

PRIOR ART DOCUMENTS Patent Documents

Published Japanese Translation of PCT No. 2002-506384

Japanese Patent (Granted) Publication No. 3,635,645

DISCLOSURE OF INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a nucleic acidextraction cartridge, a nucleic acid extraction apparatus and a nucleicacid extraction method for extracting a nucleic acid from a sample,which prevent sample contamination, and enable the complex preparationof reagents and disposal treatments that are performed before and afterthe nucleic acid extraction operation to be performed rapidly andsimply, with the extraction performed in an automated manner.

In the present invention, in light of the object described above, wellsfor storing the reagents, waste liquids and sample, and a nucleic acidseparation filter are combined in a single cartridge. By using thiscartridge, prior preparation becomes unnecessary, and a simple and rapidnucleic acid extraction method and a simple and safe disposal treatmentcan be achieved. Specifically, the aspects of the invention describedbelow are achieved.

A first aspect of the present invention is a nucleic acid extraction kithaving a container, which includes a reagent well that stores at least areagent, a sample well into which a biological sample is introduced, awaste liquid well, and a collection well in which an extracted nucleicacid is collected, and an extraction filter cartridge equipped with anextraction filter for separating and extracting a nucleic acid from thebiological sample, wherein the extraction filter cartridge is formed ina manner that enables the extraction filter cartridge to be supported onthe waste liquid well and the collection well.

A second aspect of the present invention is the nucleic acid extractionkit according to the first aspect, wherein each of the well-formingsurfaces on the container is covered with a protective film.

A third aspect of the present invention is the nucleic acid extractionkit according to the first or second aspect, wherein the containerincludes a holder for housing the extraction filter cartridge.

A fourth aspect of the present invention is the nucleic acid extractionkit according to the third aspect, wherein a member that absorbs liquidis provided on the holder.

A fifth aspect of the present invention is the nucleic acid extractionkit according to the third or fourth aspect, wherein a protective memberthat covers each of the well-forming surfaces on the container and theextraction filter cartridge is formed with the extraction filtercartridge housed in the holder.

A sixth aspect of the present invention is the nucleic acid extractionkit according to any one of the first through fifth aspects, wherein thewaste liquid well is provided with a liquid-absorbent material.

A seventh aspect of the present invention is the nucleic acid extractionkit according to any one of the first through sixth aspects, wherein awall surface that is higher than the depth to the bottom of each well isprovided around the outer periphery of the reagent cartridge, and amechanism for securing the reagent cartridge to a separate apparatus isprovided on the wall surface.

An eighth aspect of the present invention is a nucleic acid extractionmethod, which is a biological sample pretreatment method that includes afirst step of using a reagent to disrupt biological cells andextricating a nucleic acid within a human body fluid and a second stepof separating an extricated specific substance from impurities using anextraction filter, and which is a method that employs a container, whichincludes a reagent well that stores at least a reagent, a waste liquidwell and a collection well in which an extracted nucleic acid iscollected, and an extraction filter cartridge equipped with anextraction filter for separating and extracting a nucleic acid from abiological sample, wherein the nucleic acid extraction method includes:

a step of eluting a nucleic acid by mixing a biological sample and areagent, a step of supplying the solution containing the eluted nucleicacid to the extraction filter cartridge which is supported on top of thewaste liquid well, a step of retaining the nucleic acid from thesolution on the extraction filter cartridge and discarding the solutioncontaining impurities in the waste liquid well, and a step of supportingthe extraction filter cartridge on the collection well and collectingthe extracted nucleic acid.

A ninth aspect of the present invention is the nucleic acid extractionmethod according to the eighth aspect, wherein each of the well-formingsurfaces on the container is covered with a protective film, and theprotective film is pierced during nucleic acid extraction.

A tenth aspect of the present invention is the nucleic acid extractionmethod according to the eighth or ninth aspect, including a step ofremoving a solution adhered to the outer surface of the extractionfilter cartridge 12.

An eleventh aspect of the present invention is the nucleic acidextraction method according to the tenth aspect, wherein a holder inwhich the extraction filter cartridge can be installed, and a memberthat absorbs liquid and is disposed on the holder, are provided on thecontainer, and the step of removing a solution adhered to the outersurface of the extraction filter cartridge involves installing theextraction filter cartridge in the holder and wiping the solution usingthe member that absorbs liquid.

A twelfth aspect of the present invention is the nucleic acid extractionmethod according to any one of the eighth to eleventh aspects, whereinin the step of discarding the solution containing impurities in thewaste liquid well, pressure is applied using compressed air to discard aportion of the solution and retain a portion of the solution.

A thirteenth aspect of the present invention is a nucleic acidextraction apparatus, having a reagent well that stores at least areagent, a sample well into which a biological sample is introduced, awaste liquid well, a collection well in which an extracted nucleic acidis collected, and a cartridge that can be supported on the waste liquidwell and the collection well and is equipped with an extraction filterfor separating and extracting a nucleic acid from the biological sample,and also containing an extraction filter pressurization device forcausing a liquid to flow through the extraction filter, a device formoving the cartridge, and a dispensing device for moving liquid reagentsand samples.

A fourteenth aspect of the present invention is the nucleic acidextraction apparatus according to the thirteenth aspect, wherein thereagent well, the sample well, the waste liquid well and the collectionwell are formed within a single container, and the container isinstalled on a detachable fixed base.

A fifteenth aspect of the present invention is the nucleic acidextraction apparatus according to the thirteenth or fourteenth aspect,wherein the extraction filter pressurization device uses a compressedair supply mechanism to supply compressed air from above the cartridge,thereby causing a liquid to flow through the extraction filter.

A sixteenth aspect of the present invention is the nucleic acidextraction apparatus according to the fifteenth aspect, wherein thecompressed air supply mechanism is equipped with an electronic controlregulator that enables arbitrary setting of the compressed air appliedpressure.

A seventeenth aspect of the present invention is the nucleic acidextraction apparatus according to any one of the thirteenth to sixteenthaspects, wherein the apparatus is provided with a holder for housing thecartridge.

An eighteenth aspect of the present invention is the nucleic acidextraction apparatus according to any one of the thirteenth toseventeenth aspects, wherein the dispensing device employs a dispensingpipette composed of a dispensing nozzle equipped with aspiration anddischarge mechanisms, and a detachable dispensing pipette tip that isprovided at the tip of the dispensing nozzle.

A nineteenth aspect of the present invention is a nucleic acidextraction method that uses the nucleic acid extraction apparatusaccording to any one of the thirteenth to eighteenth aspects, whereinthe nucleic acid extraction method includes a step of eluting thenucleic acid using the dispensing device to add a biological sample thathas been introduced into the sample well to a well containing a reagentfor eluting the nucleic acid, a step of adding the solution containingthe eluted nucleic acid to the cartridge, which has been supported abovethe waste liquid well using the device for moving the cartridge, usingthe dispensing device, a step of retaining the nucleic acid from thesolution and discarding the solution containing impurities into thewaste liquid well using the extraction filter pressurization device, anda step of collecting the extracted nucleic acid from the cartridge,which has been supported above the collection well using the device formoving the cartridge.

A twentieth aspect of the present invention is the nucleic acidextraction method according to the nineteenth aspect, wherein each ofthe well-forming surfaces is covered with a protective film, and theprotective film is pierced during nucleic acid extraction.

A twenty first aspect of the present invention described in claim 7 or 8is the nucleic acid extraction method according to the nineteenth ortwentieth aspect, wherein in the step of discarding the solutioncontaining impurities into the waste liquid well, pressure is appliedusing compressed air to discard a portion of the solution containingimpurities and retain a portion of the solution.

A twenty second aspect of the present invention is the nucleic acidextraction method according to any one of the nineteenth to twenty firstaspects, wherein a holder in which the cartridge can be installed, and amember that absorbs liquid and is disposed on the holder, are providedin the nucleic acid extraction apparatus, and a step of removing asolution adhered to the outer surface of the extraction filter cartridgeis performed by installing the extraction filter cartridge in the holderand wiping the solution using the member that absorbs liquid.

A twenty third aspect of the present invention is the nucleic acidextraction method according to any one of the nineteenth to twentysecond aspects, wherein following the step of collecting the nucleicacid, residual biological sample liquid left in the sample well isaspirated and transported to the waste liquid well.

Effect of the Invention

According to the present invention, by supporting an extraction filterfor extracting a nucleic acid directly on a reagent container in such amanner that enables extraction to be performed, and then integratingthese components into a single unit, the instruments used in a singlenucleic acid extraction operation can be simplified, and the operationsrequiring human input can be simplified. Moreover, by providing, withinthe container, a waste liquid well into which reagents can be discarded,reagent preparation and waste liquid treatment following the nucleicacid collection can be simplified in those cases where an automatednucleic acid extractor is used, enabling the nucleic acid extraction tobe performed rapidly, simply, and with no contamination.

Moreover, according to the nucleic acid extraction apparatus of thepresent invention, reagent preparation and waste liquid treatmentfollowing the nucleic acid collection can be simplified in those caseswhere an automated nucleic acid extractor is used, enabling the nucleicacid extraction to be performed rapidly, simply, and with nocontamination. More specifically, by installing a cartridge equippedwith an extraction filter directly on top of the waste liquid well andcollection well, and then performing the separation and extractionoperations using these structural elements, contamination caused bysolution splashing due to extraction filter pressurization oroverturning of the extraction filter members can be eliminated, enablingthe extraction operation to be simplified.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view illustrating the waste liquid side of themain body of a reagent cartridge according to the present invention.

FIG. 2 is a schematic view of the main body of the reagent cartridgeillustrated in FIG. 1.

FIG. 3 is a schematic view of an extraction filter cartridge.

FIG. 4 is a perspective view of a nucleic acid extraction apparatusaccording to the present invention.

FIG. 5 is a flowchart describing the extraction of a nucleic acid from asample using the nucleic acid extraction method of the presentinvention.

FIG. 6 is a perspective view for describing the nucleic acid extractionmethod according to the present invention, and illustrates the operationof the apparatus.

FIG. 7 is a perspective view for describing the nucleic acid extractionmethod according to the present invention, and illustrates the operationof the apparatus.

FIG. 8 is a perspective view for describing the nucleic acid extractionmethod according to the present invention, and illustrates the operationof the apparatus.

BEST MODE FOR CARRYING OUT THE INVENTION 1. Nucleic Acid ExtractionApparatus

The present invention is described below based on the drawings. FIG. 1is a perspective view of one embodiment of a reagent cartridge accordingto the present invention. FIG. 2 is a plan view illustrating each of thewell-forming surfaces of the container of the reagent cartridge. Asdescribed below, by supplying a biological sample to the sample well ofa reagent cartridge 100 and then installing the cartridge in the nucleicacid extraction apparatus, the nucleic acid extraction operation can beperformed in an automated manner. A reagent container 1 that constitutesthe reagent cartridge of the present invention is composed of a basematerial that forms the container, in which are formed reagent wells 2to 8, a waste liquid well 10 in which unnecessary solutions separatedduring the extraction process are discarded, a collection well 11 forcollecting the extracted nucleic acid, and a sample well 13 into whichthe biological sample is introduced. Each of the reagent wells containsa liquid, such as a solvent for dissolving biological matter such ascell membranes or the like, a diluent for diluting solutions, an eluentfor eluting the nucleic acid from the carrier, or a washing liquid forwashing away unnecessary solutions, although the solutions containedwithin the wells are not limited to these types of liquids. Moreover, anucleic acid extraction kit according to the present invention is alsoprovided with an extraction filter cartridge 12 such as that illustratedin FIG. 3, which is independent from the reagent cartridge main body(the reagent container). The reagent cartridge of the present inventionis secured to a nucleic acid extraction apparatus 200 such as thatillustrated in FIG. 4, enabling an extraction operation using adispensing pipette to be performed mechanically.

Each of the structural elements of the apparatus are described below.The apparatus and structural elements of the present invention are notlimited to the locations and configurations illustrated in the drawings,and may also be combined with other structural elements, provided theactions and effects of the present invention are not impaired.

<Reagent Cartridge Main Body>

The shape of the reagent container 1 may be any shape that can besecured to the nucleic acid extraction apparatus for subsequent use.Examples of the method used for securing the container include providinglugs 20 on the reagent cartridge which can be hooked onto the apparatusto secure the cartridge, although this is not the only possible method.As illustrated in FIG. 2, the reagent container 1 may be formed as asubstantially rectangular shape having a region in which the reagentwells are arrayed, a region in which the waste liquid well is formed, aregion in which the collection well is formed, and a region in which thesample well is formed. Further, as illustrated in FIG. 1, a wall surfaceor footing that is higher than the depth to the bottom of each well ispreferably provided around the outer periphery of the reagent container,thus forming a stand-alone structure. By employing such a structure,safety can be improved during operation, for example if the container isleft standing temporarily by a user, meaning blood contamination or thelike caused by the container overturning can be prevented. Moreover, themechanism for securing the container to the apparatus, such as the lugs20 mentioned above, may be provided on the wall surface.

The waste liquid well 10 and the collection well 11 each has a shapethat enables the extraction filter cartridge 12 to be supported thereon,and the extraction filter cartridge 12 can be secured to both of thesewells in a detachable manner. In the present invention, “a shape thatenables the extraction filter cartridge to be supported thereon” meansthat the reagent container secures the extraction filter cartridge inplace, preventing overturning or the like. For example, by matching theexternal shape of the extraction filter cartridge with the shape of theopening of each well (or a portion of the shape), a shape that enablesthe extraction filter cartridge to be supported thereon can be achieved.

As described below, during the extraction process, the extraction filtercartridge 12 is moved from the waste liquid well 10 to the collectionwell 11, and therefore the waste liquid well and the collection well arepreferably positioned adjacently. By minimizing the movement distance,contamination of the top of the cartridge can be prevented. Further, thereagent wells are mainly used for performing dispensing operations tothe extraction filter cartridge when the cartridge is positioned abovethe waste liquid well, and therefore the waste liquid well and theregion in which the reagent wells are formed are also preferablyadjacent. This enables contamination to be prevented and also enablesthe time of the operations to be shortened.

Further, an extraction filter cartridge holder 18 for housing theextraction filter cartridge 12 is preferably provided in the reagentcontainer 1. Because this enables the reagent cartridge to be installedin the apparatus with the extraction filter cartridge already supportedin the holder 18, handling is simplified. Furthermore, as describedbelow, by integrating the extraction filter cartridge 12 with thereagent container 1, both the container and the extraction filtercartridge 12 can be covered with a protective member, thereby preventingincorporation of foreign matter or other contamination.

A member that absorbs liquid may be provided in the bottom of theextraction filter cartridge holder 18. This member is positioned so thatwhen the extraction filter cartridge 12 is positioned in the holder, aportion of the outer surface of the extraction filter cartridge mainbody 12 a, and at least the tip thereof, contacts the member.Immediately following those steps in which there is a possibility of asolution adhering to the outer surface of the extraction filtercartridge, by moving the extraction filter cartridge from the positionabove the waste liquid well back to the extraction filter cartridgeholder, the tip of the extraction filter cartridge makes contact withthe member that absorbs liquid, enabling any adhered solution to beabsorbed and removed, thereby preventing contamination by the adheredwaste liquid. Examples of materials that may be used for the member thatabsorbs liquid include absorbent materials such as filter paper orsponges.

There are no particular limitations on the material used for forming thereagent container 1 provided it has no effect on the sample and reagentsand the like, but using a resin material containing any one ofpolypropylene, polycarbonate or acrylic enables favorable visible lighttransmittance to be achieved, thus enabling the state of the varioussolutions to be confirmed. Examples of the polypropylene includehomopolypropylene and random copolymers of polypropylene andpolyethylene. Examples of the acrylic include poly(methyl methacrylate),and copolymers of methyl methacrylate with another monomer such asanother methacrylate ester, an acrylate ester or styrene. Further, usingthese resin materials enables better tip heat resistance and strength tobe obtained.

In the case of a resin material, examples of methods that may be usedfor forming the container 1 include various resin molding methods suchas injection molding and vacuum molding methods, as well as machinecutting methods.

A member that absorbs waste liquid is preferably installed in the wasteliquid well 10. By installing this absorbent member, moisture thatexists inside the waste liquid well is absorbed, meaning nocontamination occurs if the waste liquid well 10 is overturned, andtherefore any potential deleterious effects on humans can be prevented.Further, by using an absorbent material that expands in volume uponmoisture absorption to absorb the waste liquid, the absorbent materialcan expand inside the waste liquid well 10, thereby preventingcontamination. Examples of the absorbent member include porous materialstypified by sponges, and absorbent polymers typified by sodiumpolyacrylate. Of these, sponges and the like can be altered in size sothat the outer diameter of the sponge in its natural state prior toinsertion in the waste liquid well is the same as, or slightly largerthan, the internal diameter of the waste liquid well, thereby enablingthe sponge to be secured in a predetermined position inside the wasteliquid well.

<Extraction Filter Cartridge>

The reagent cartridge 100 of the present invention is provided with theextraction filter cartridge 12, which is equipped with an extractionfilter for separating and extracting the nucleic acid from thebiological sample. The extraction filter cartridge 12 includes at leastan extraction filter 12 b secured to the extraction filter cartridgemain body 12 a.

One example of the extraction filter cartridge is illustrated in FIG. 3.In the extraction filter cartridge illustrated in FIG. 3, a filtermember 12 b for adsorbing the nucleic acid is supported in the center ofa cylindrical main body 12 a that is open at the upper edge, and afilter support 12 c is provided directly beneath the filter member 12 bto prevent deformation of the filter member 12 b. The lower portion ofthe cylindrical main body 12 a located beneath the filter member isformed in a funnel shape, and a nozzle-shaped discharge port 12 d thatextends for a predetermined length is formed in the center of the lowerportion of the main body 12 a. The sample solution, washing solution andcollection solution described below are dispensed into the upper openingof the extraction filter cartridge, and compressed air is thenintroduced from the upper opening, thereby forcing each of the solutionsthrough the filter member 12 b and the filter support 12 c, and then outof the discharge port 12 d and into the waste liquid well 10 orcollection well 11 described below, where the solution is eitherdiscarded or collected.

The filter member 12 b of the above-mentioned extraction filtercartridge 12 is formed from a membrane of a porous material that adsorbsthe nucleic acid, and is configured so that the nucleic acid is adsorbedand retained during washing with the washing liquid, but is thenreleased as the nucleic acid-adsorbing power of the membrane is weakenedduring collection using the collection liquid. In the present invention,this porous material includes materials that are combined with fibrousmaterials such as glass wool. There are no particular limitations on thematerial of the filter, provided it is capable of adsorbing biologicalmatter in the presence of organic material, although the use of porousmaterials containing introduced hydroxyl groups as hydrophilic groups ispreferable. Silica or other materials having silica combined therewithcan be used particularly favorably. The extraction filter cartridge mainbody 12 a can be formed using the same materials and formation methodsas the reagent container 1.

The filter support 12 c may be laminated beneath and/or above the filtermember 12 b. The filter member 12 b flexes upon pressurization with thepressurization mechanism, resulting in the danger of undesirable partialsolution leakage, but by laminating a comparatively rigid filter support12 c, such leakage can be prevented. In terms of the method ofpreparation, the use of a filter prepared by hardening resin particlesby baking is preferable, although the invention is not limited to thisparticular method, and any filter having pores that enable the materialrelated to the reaction to be retained while the solution passes throughthe filter can be used. There are no particular limitations on thematerial for the filter, provided it can be formed in a porous statethat enables the solutions to pass through the filter.

<Protective Member>

In order to prevent incorporation of foreign matter or othercontamination, a protective member is preferably provided on top of thereagent container 1. In the example of the present invention illustratedin FIG. 1, the open surfaces of the reagent wells 2 to 9 are sealed witha protective film 14 a. The protective film 14 a covers at least theopen surfaces of each of the reagent wells, and a separate film may beprovided for each reagent well, a plurality of reagent wells may becovered with a single seal, or the entire region in which the reagentwells are formed may be covered with a single seal.

Moreover, by forming the protective film 14 a from a material that has alevel of strength that can be pierced by the tip of the dispensingpipette (the dispensing pipette tip) used during the dispensing process,the dispensing pipette can simply be pierced through the protective film14 a from above to extract the reagents from their respective wellsduring the nucleic acid extraction operation, and therefore there is noneed to remove the protective film 14 a, and no possibility of reagentcontamination.

In order to enable storage of the extraction filter cartridge 12, asecond protective member is preferably provided on top of the protectivefilm 14 a. In the example of the present invention illustrated in FIG.1, all of the well-forming surfaces of the reagent container 1,including the sample well 13, the waste liquid well 10, and theextraction filter cartridge holder 18 are covered and sealed withanother protective film 14 b. Accordingly, introduction of foreignmatter into these wells or other contamination can be prevented. Byremoving the second protective member when the reagent container isinstalled in the nucleic acid extraction apparatus, operations such asthe filling of the sample well 13 with a biological material such asblood can be performed.

By using the above type of structure including a two-stage protectivemember, protection can be divided between a region that is openedimmediately prior to installation within the apparatus, and a regionthat includes the reagent wells and the like which remains sealed untilimmediately prior to use. In other words, even after the protective film14 b has been removed, because the protective film 14 a still exists onthe reagent wells until immediately prior to use, safe storage can beachieved with no risk of contamination.

The protective films 14 a and 14 b can use any film shape that does noteffect the sample or the reagents or the like, and examples includeplastic films such as various polypropylenes or polyesters such aspolyethylene terephthalate and polynaphthalate, as well as films ofmetal foil formed from aluminum or the like. The protective film 14 a isbonded so as to close the openings of each reagent well, thereby sealingeach reagent well. In the case of a plastic film, sealing can beperformed by fusing the plastic using heat or a laser. Further, using amaterial that exhibits transparency for the protective film offers theadvantage that, following heat fusion, the sealing properties can beconfirmed by visual inspection of the fused surface. The expression thatthe film “exhibits transparency” means that the film must transmit atleast a portion of the visible light spectrum, so that the sealingproperties can be confirmed visually. The protective film may have alaminated structure. For example, one side of the film may have anexposed plastic film that acts as the fusion surface, while the otherside may be formed from a layer of an inorganic material such as SiO₂,thereby improving the sealing properties.

In another embodiment of the protective member, besides the protectivefilm, a portion of the well-forming surfaces of the reagent container 1including the sample well 13 of the reagent cartridge main body may beprovided with a structure that can be opened and closed with a singletouch, such as a snap-fit structure. By also employing a similarsnap-fit structure on the waste liquid well 10 for when the cartridge isdiscarded following use, splashing of the sample can be prevented, andthe reagent cartridge main body 1 can be disposed of in a safe manner.

<Dispensing Pipette>

In order to prevent contamination, the dispensing pipette used todispense the reagents is preferably discarded, together with the reagentcontainer 1, following a single extraction of nucleic acid. Accordingly,in the nucleic acid extraction apparatus of the present inventionillustrated in FIG. 4, by providing a dispensing nozzle 16 equipped withliquid aspiration and discharge mechanisms separately from a dispensingpipette tip 17 that is detachable from the dispensing nozzle, only thedispensing pipette tip 17 need be discarded.

The dispensing pipette tips are preferably installed in the apparatus ina configuration wherein a plurality of the dispensing pipette tips 17 ato 17 d which are each used in a single extraction operation of nucleicacid are stored in a dispensing pipette tip rack 21 capable of storingthe plurality of dispensing pipette tips. By using this type ofstructure, when operations have been completed, the dispensing pipettetips can be discarded by simply discarding the entire dispensing pipettetip rack, thereby simplifying the disposal operation.

In the dispensing pipette tip rack 21, a wall surface or footing that ishigher than the depth to the bottom of the dispensing pipette tipstorage portion is preferably provided around the outer periphery of thedispensing pipette tip rack, thus forming a stand-alone structure. Byemploying such a structure, safety can be improved during operation, forexample if the user leaves the dispensing pipette tip rack standingtemporarily, meaning blood contamination or the like caused by the rackoverturning can be prevented. Furthermore, a mechanism for securing thedispensing pipette tip rack to the apparatus, such as the lugs describedabove, may be provided on the wall surface of the stand-alone structure.Moreover, by providing a lid on the dispensing pipette tip storageportion of the dispensing pipette tip rack, contamination by foreignmatter prior to use can be prevented, and contamination caused bysplashing of solution adhered to the dispensing pipette tips followingextraction can also be prevented.

<Nucleic Acid Extraction Apparatus>

FIG. 4 is a perspective view illustrating the structural elements of anucleic acid extraction apparatus 200 according to the presentinvention, which includes the reagent cartridge 100 described above. Thenucleic acid extraction apparatus according to the present inventionincludes at least an extraction filter pressurization device for causingthe sample liquid to flow through the extraction filter, a device formoving the extraction filter cartridge, and a dispensing device formoving liquid reagents and samples. The reagent cartridge may beinstalled on a detachable fixed base prior to use. In the nucleic acidextraction apparatus of the present invention, because the reagentcontainer 1 containing each of the wells, and the extraction filtercartridge 12 can be installed as a single cartridge, the operationsfollowing insertion of the cartridge can be automated, enabling theextraction process to be simplified.

However, the apparatus is not limited to the reagent cartridgeconfiguration described above, and in the nucleic acid extractionapparatus of the present invention, by positioning the extraction filtercartridge 12 directly on the waste liquid well 10 and the collectionwell 11, and performing the separation and extraction operations usingeach of the structural elements mentioned above, splashing of thesolution during pressurization of the extraction filter andcontamination caused by overturning of the extraction filter member canbe eliminated, and the extraction operation can be simplified. Each ofthe structural elements is described below in further detail.

The extraction filter pressurization device is a structural element inwhich, by using a compressed air supply mechanism 15 provided downstreamfrom an air pump and connected to an electronic control regulator topressurize and then project air or an inert gas such as nitrogen ontothe top of the extraction filter cartridge 12, a portion of the solutionintroduced into the extraction filter cartridge is forced through theextraction filter, thereby achieving separation. By providing theelectronic control regulator, the compressed air applied pressure can beset as desired.

The device for moving the extraction filter cartridge is used for movingthe extraction filter cartridge 12 from the holder 18 to a positionabove the waste liquid well 10 or the collection well 11. In the nucleicacid extraction apparatus illustrated in FIG. 4, an extraction filtercartridge support mechanism 19 that is able to support a flange formedon the extraction filter cartridge 12 is provided beneath the compressedair supply mechanism 15, and the pressurization step can be performed bysimply using a lift mechanism provided with the movement mechanism tolower the compressed air supply mechanism following completion of themovement.

A dispensing pipette can be used as the dispensing device for dispensingthe liquid reagents and samples stored in the various wells. In thenucleic acid extraction apparatus illustrated in FIG. 4, the dispensingdevice is composed of the dispensing nozzle 16 equipped with liquidaspiration and discharge mechanisms, and the dispensing pipette tip 17,which is the detachable end portion of the dispensing pipette. Byproviding a plurality of dispensing pipette tips 17 a to 17 d, thenucleic acid extraction operation can be performed with a singledispensing nozzle, by changing the dispensing pipette tip for eachdifferent solution. In this case, a configuration is preferably used inwhich the dispensing nozzle can be moved to a position above thedispensing pipette tip, and the tip then attached or detachedmechanically. The dispensing pipette tips can be stored in a dedicatedcartridge, which can be installed on the apparatus either at the sametime as, or separately from, the reagent cartridge.

By providing a movement device for the compressed air supply mechanism15, the extraction filter cartridge support mechanism 19 and thedispensing nozzle 16, the extraction process following installation ofthe reagent cartridge can be automated. The movement device may use acommon movement mechanism, or may be provided with a series ofindividual movement mechanisms.

Further, the mount (not shown in the drawings) for installing thereagent cartridge 100 on the nucleic acid extraction apparatus may beprovided with a heating block. This heating block may be used to heat aportion of the wells of the reagent container 1, thereby acceleratingsome of the extraction steps.

2. Nucleic Acid Extraction Method

A nucleic acid extraction from blood is described below as an example ofthe nucleic acid extraction method of the present invention, withreference to steps S1 to S14 of the flowchart of FIG. 5, whichillustrates the nucleic acid extraction method of the present invention,and the perspective views of FIG. 6 to FIG. 8, which illustrate theoperation of the nucleic acid extraction apparatus during the extractionprocess. Examples of other biological samples besides blood that may besubjected to nucleic acid extraction using the same method includevarious bodily fluids such as plasma, serum, semen and lymphatic fluid,various tissues such as skin, hair and muscle tissue, and variousexcreted substances such as feces and urine obtained from a targetliving organism (or the corpse of a living organism), as well as othersamples derived from the above samples. The basic configuration of thenucleic acid extraction method is the same as the widely used BOOMmethod. The BOOM method is a nucleic acid extraction technique thatcombines a chaotropic agent with a solid-phase silica or the like, andutilizes the adsorption of the nucleic acid onto the silica surface inthe presence of chaotropic ions.

First, the collected blood is injected into the sample well 13 of thereagent cartridge 100, from which the second protective member (theprotective film 14 b) has been removed. By then installing the reagentcartridge in the nucleic acid extraction apparatus, the operationsdescribed below can be controlled electronically and performed in anautomated manner. By adopting such a configuration, dispensingoperations can be performed using the reagent cartridge main body 1, andthe extraction of the biological matter can be automated. In those caseswhere a dispensing pipette tip cartridge is to be used, this cartridgeis also installed on the extraction apparatus.

<Nucleic Acid Extraction Process>

The extraction filter cartridge 12 is moved to a position above thewaste liquid well 10 using the device for moving the extraction filtercartridge (Si). The blood added to the sample well 13 is then aspiratedusing the dispensing pipette (S2). Subsequently, the blood istransported to the solvent within a reagent well and mixed with thesolvent to elute the nucleic acid (DNA) from the blood (S3). At thistime, by using the tip of the dispensing pipette to pierce theprotective film 14 a on top of the reagent well before dispensing theblood, the sealing properties of the reagent are maintained until thetime of operation, enabling the extraction operation to be performedwithout any contamination or degradation. The dispensing pipette is usedto add the blood to the reagent well 5, which is filled with achaotropic agent (solvent) that dissolves the biological matter (FIG.6). The solvent and the blood are mixed thoroughly inside the reagentwell by repeatedly discharging and aspirating the mixed liquid using thepipette, thereby dissolving the biological matter.

Next, the dissolved solution is transported to the extraction filtercartridge 12 positioned above the waste liquid well 10 using thedispensing nozzle 16 and the dispensing pipette tip 17, and the solutionis then added to the filter member 12 b (S4). The nucleic acid elutedfrom the white blood cells in the blood is adsorbed to the filter member12 b (FIG. 7).

Subsequently, the compressed nozzle 15 is moved above the extractionfilter and lowered into place, and compressed air is then used to forcethe solution containing the non-adsorbed impurities into the wasteliquid well as a waste liquid (S5, FIG. 8). At this point, if all of thesolution is to be removed from the filter member 12 b, then a pressurethat is larger than the pressure required to force the solution throughthe filter must be applied. However, this increased pressure means thatthere is an extremely high probability of splashing or foaming of thesolution, causing contamination of the inside of the apparatus or theoperating environment. Accordingly, a lower pressure that does not causeall of the solution to pass through the filter is preferably applied. Inother words, in the air pressurization step described above, a portionof the solution containing impurities is retained in the vicinity of thedischarge port 12 d of the extraction filter cartridge, but by repeatingthe nucleic acid washing step described below, any residual impuritiescan be removed. Alternatively, a plurality of elution steps may beprovided. In other words, the pipette may be used to aspirate a solventfrom another reagent well 4 filled with a chaotropic agent (solvent) andthen discharge this solvent onto the extraction filter (S6). Compressedair is then used to force the solution containing the impurities thathave not been adsorbed to the filter into the waste liquid well as awaste liquid (S7). By including a plurality of elution steps in thismanner, the nucleic acid may be eluted using a pressure that does notresult in contamination of the apparatus.

<Nucleic Acid Washing Process>

The washing process for the filter having the adsorbed nucleic acid isperformed by aspirating a washing liquid from the reagent well 3containing the washing liquid using the dispensing device, and thenadding the washing liquid to the extraction filter cartridge 12 (S8).Compressed air from the extraction filter pressurization device is thenused to force the impurities that have not been adsorbed or bound to thefilter into the waste liquid well 10 (S9). The adsorbed nucleic acid iswashed repeatedly by repeating this washing process using the washingliquid in the reagent well 7 (S10, S11). In those cases where aplurality of dispensing pipettes (dispensing pipette tips 17) areprovided, the pipette tip may be changed each time a different reagentis used during the washing process described above and the collectionprocess described below.

<Collection Process>

The collection well 11 is used for collection of the nucleic acid.First, the extraction filter cartridge 12 is transported by the devicefor moving the extraction filter cartridge to a position above thecollection well 11, in order to enable collection of the nucleic acid inthe clean collection well 11 (S12). The eluent in the elution reagentwell 9 is then added to the extraction filter cartridge 12 from thedispensing pipette tip 17 of the dispensing device (S13). Then, in asimilar manner to that described above, compressed air from thecompressed air nozzle 15 is used to collect the nucleic acid eluted fromthe extraction filter cartridge 12, together with the eluent, in thecollection well 11 (S14). The diluent of the regent well 8 may be usedto alter the concentration of the collected solution.

Between the nucleic acid extraction process and the collection process,when the extraction filter cartridge is moved from the collection wellor the waste liquid well, a step may be provided for removing solutionadhered to the outer surface of the extraction filter cartridge 12. Bybringing at least a portion of the outer surface of the extractionfilter cartridge 12, and particularly the tip of the cartridge in thosecases where the cartridge has a funnel-like shape, as the tip isparticularly prone to solution adherence, into contact with a memberthat absorbs liquid, the adhered solution can be removed by wiping. Thisenables the prevention of contamination caused by adhered waste liquid.As described above, this process can be performed by moving theextraction filter cartridge to the extraction filter cartridge holder 18which is provided with a member that absorbs liquid, and bringing theextraction filter cartridge into contact with the member that absorbsliquid, thereby absorbing and removing the adhered solution. Byemploying this type of configuration, a separate mechanism for removingadhered solutions need not be provided, and contamination caused bymovement of the extraction filter cartridge can be prevented.

Further, in those cases where excess blood exists in the sample well, astep may be provided for aspirating the excess blood using the pipette,and discarding the blood in the waste liquid well 10. If an absorbentmaterial is provided in the waste liquid well, then no splashing of theliquid occurs, and the reagent container 1 can be discarded without anycontamination of the apparatus by the excess blood.

Following completion of this series of automated nucleic acid extractionoperations, the nucleic acid may be collected from the collection well11 using a pipette tip, or may be placed in an automated nucleic acidsequencing apparatus, and transported immediately and automatically to adetection system. By storing the extraction filter cartridge 12 in apredetermined location on the reagent container 1 (either in the holder18 or above the supporting waste liquid well 10), the entire structurecan be discarded safely and simply as medical waste.

As described above, in the present invention, because an integratednucleic acid extraction cartridge is provided as a nucleic acidextraction kit containing the reagent container 1 and the extractionfilter cartridge, once the biological sample has been added to thesample well and the extraction kit has been installed in the nucleicacid extraction apparatus, the nucleic acid extraction operations can becompletely automated. Further, because the waste liquid well is formedwithin the cartridge, no waste liquid is left in the apparatus followingthe nucleic acid extraction, and treatment of the waste liquid can besimplified. Accordingly, there is no danger of contamination of theapparatus with waste liquid. Furthermore, at least a portion of thewells formed inside the reagent cartridge main body are formed fromplastic film, and because the reagents are provided within an integratedcartridge, handling is simple. Moreover, because the cartridge is asingle integrated unit, disposal can also be performed safely.

DESCRIPTION OF THE REFERENCE SIGNS

-   1: Reagent cartridge main body (reagent container)-   3: Reagent well (washing liquid)-   4: Reagent well (solvent)-   5: Reagent well (solvent)-   7: Reagent well (washing liquid)-   8: Reagent well (diluent)-   9: Reagent well (eluent)-   10: Waste liquid well-   11: Collection well-   12: Extraction filter cartridge-   12 a: Extraction filter cartridge main body-   12 b: Filter member-   12 c: Filter support-   12 d: Discharge port-   13: Sample well-   14: Protective member-   14 a: First protective member (protective film)-   14 b: Second protective member (protective film)-   15: Compressed air nozzle-   16: Dispensing nozzle-   17: Dispensing pipette tip-   18: Holder-   19: Extraction filter cartridge support mechanism-   20: Lug-   21: Dispensing pipette tip rack-   100: Reagent cartridge-   200: Nucleic acid extraction apparatus

1. A nucleic acid extraction kit, having a container comprising areagent well therein that stores at least a reagent, a sample well intowhich a biological sample is introduced, a waste liquid well, and acollection well in which an extracted nucleic acid is collected and anextraction filter cartridge comprising an extraction filter forseparating and extracting a nucleic acid from the biological sample,wherein the extraction filter cartridge is formed in a manner thatenables the extraction filter cartridge to be supported on the wasteliquid well and the collection well.
 2. The nucleic acid extraction kitaccording to claim 1, wherein each well-forming surface on the containeris covered with a protective film.
 3. The nucleic acid extraction kitaccording to claim 2, wherein the protective film is formed from amaterial that can be pierced.
 4. The nucleic acid extraction kitaccording to claim 1, wherein the container comprises a holder forhousing the extraction filter cartridge in the container.
 5. The nucleicacid extraction kit according to claim 4, wherein a member that absorbsliquid is provided on the holder.
 6. The nucleic acid extraction kitaccording to claim 1, wherein the waste liquid well comprises aliquid-absorbent material.
 7. The nucleic acid extraction kit accordingto claim 1, wherein a wall surface that is higher than a depth to abottom of each well is provided around an outer periphery of the reagentcartridge, and the container comprises a mechanism on the wall surfaceof the container for securing the container to a separate apparatus. 8.A nucleic acid extraction method, which is a biological samplepretreatment method comprising a first step of using a reagent todisrupt biological cells and extricating a nucleic acid within a humanbody fluid, and a second step of separating an extricated specificsubstance from impurities using an extraction filter, and which is amethod that employs a container comprising a reagent well therein thatstores at least a reagent, a waste liquid well and a collection well inwhich an extracted nucleic acid is collected, and an extraction filtercartridge comprising an extraction filter for separating and extractinga nucleic acid from a biological sample, and the nucleic acid extractionmethod comprises: a step of collecting a biological sample from a samplewell in the container with a dispensing device, a step of eluting anucleic acid by mixing the biological sample and the reagent, a step ofadding a solution containing the eluted nucleic acid to the extractionfilter cartridge which is supported on top of the waste liquid well, astep of retaining the nucleic acid from the solution on the extractionfilter cartridge and discarding a solution containing impurities in thewaste liquid well, and a step of supporting the extraction filtercartridge on the collection well and collecting the extracted nucleicacid into the collection well.
 9. The nucleic acid extraction methodaccording to claim 8, wherein each well-forming surface on the containeris covered with a protective film, and the protective film is piercedduring nucleic acid extraction.
 10. The nucleic acid extraction methodaccording to claim 8, further comprising a step of removing a solutionadhered to an outer surface of the extraction filter cartridge when theextraction filter cartridge is moved from the collection well or thewaste liquid well, wherein a holder in which the extraction filtercartridge can be installed, and a member that absorbs liquid and isdisposed on the holder are provided in the container, and the step ofremoving the solution adhered to the outer surface of the extractionfilter cartridge comprises installing the extraction filter cartridge inthe holder and wiping the solution using the member that absorbs liquid.11. The nucleic acid extraction method according to claim 8, wherein inthe step of discarding the solution containing impurities in the wasteliquid well, applying pressure to the extraction filter using compressedair to discard a portion of the solution into the waste liquid well andretain a portion of the solution on the filter of the extraction filtercartridge.
 12. A nucleic acid extraction apparatus, comprising a reagentwell therein that stores at least a reagent, a sample well into which abiological sample is introduced, a waste liquid well, a collection wellin which an extracted nucleic acid is collected, a cartridge that can besupported on the waste liquid well and the collection well and comprisesan extraction filter for separating and extracting a nucleic acid from abiological sample, and the nucleic acid extraction apparatus comprisingan extraction filter pressurization device for causing a liquid to flowthrough the extraction filter, a device for moving the cartridge, and adispensing device for moving liquid reagents and samples.
 13. Thenucleic acid extraction apparatus according to claim 12, wherein thereagent well, the sample well, the waste liquid well and the collectionwell are formed within a single container, and the container isinstalled on a detachable fixed base.
 14. The nucleic acid extractionapparatus according to claim 12, wherein the extraction filterpressurization device uses a compressed air supply mechanism to supplycompressed air from above the cartridge, thereby causing a liquid toflow through the extraction filter.
 15. The nucleic acid extractionapparatus according to claim 14, wherein the compressed air supplymechanism comprises an electronic control regulator that enablesarbitrary setting of the compressed air applied pressure.
 16. Thenucleic acid extraction apparatus according to claim 12, wherein theapparatus comprises a holder for housing the cartridge in the container.17. The nucleic acid extraction apparatus according to claim 12, whereinthe dispensing device employs a dispensing pipette composed of adispensing nozzle equipped with aspiration and discharge mechanisms, anda detachable dispensing pipette tip that is provided at the tip of thedispensing nozzle.
 18. A nucleic acid extraction method that uses thenucleic acid extraction apparatus according to claim 12, the nucleicacid extraction method comprising: a step of collecting a biologicalsample from a sample well in the container with a dispensing device, astep of eluting the nucleic acid by mixing the biological sample and thereagent using the dispensing device to add a biological sample that hasbeen introduced into the sample well to the reagent for eluting thenucleic acid, a step of adding a solution containing the eluted nucleicacid to the cartridge, which has been supported above the waste liquidwell using the device for moving the cartridge, using the dispensingdevice, a step of retaining the nucleic acid from the solution on theextraction filter-and discarding a solution comprising impurities intothe waste liquid well using the extraction filter pressurization device,and a step of collecting the extracted nucleic acid into the collectionwell from the cartridge, which has been supported above the collectionwell using the device for moving the cartridge.
 19. The nucleic acidextraction method according to claim 18, wherein each well-formingsurface is covered with a protective film, and the protective film ispierced during nucleic acid extraction.
 20. The nucleic acid extractionmethod according to claim 18, wherein in the step of discarding thesolution comprising impurities into the waste liquid well, pressure isapplied to the extraction filter using compressed air to discard aportion of the solution comprising impurities into the waste liquid welland retain a portion of the solution on the filter of the cartridge. 21.The nucleic acid extraction method according to claim 18, furthercomprising a step of removing the solution adhered to outer surface ofthe cartridge when the cartridge is moved from collection well or thewaste liquid well, wherein a holder in which the cartridge can beinstalled, and a member that absorbs liquid and is disposed on theholder, are provided in the container, and the step of removing thesolution adhered to the outer surface of the cartridge comprisesinstalling the cartridge in the holder and wiping the solution using themember that absorbs liquid.
 22. The nucleic acid extraction methodaccording to claim 18, wherein following the step of collecting theextracted nucleic acid aspirating, residual biological sample liquidleft in the sample well and transporting to the waste liquid well.